Medium additives and media for culturing animal cells

ABSTRACT

An object of the present invention is to provide a medium supplement for animal cell culture and an animal cell culture medium. The present invention relates to a medium supplement for animal cell culture comprising sericin or a sericin derivative and an animal cell culture medium comprising at least said medium supplement and a basal medium composition.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a medium supplement for animalcell culture and an animal cell culture medium containing the same.

[0003] 2. Background Art

[0004] In the field of life science, it has recently become important toproduce useful substances in an industrial scale by mass culture ofanimal cells that produce a substance of interest, using cell culturetechnology or tissue culture technology. Animal cells of interest arecultured in this way with a culture medium which contains a basal mediumwhich comprises amino acids, vitamins, inorganic salts, sugars and thelike and an animal cell growth factor. As an animal cell growth factor,a serum component, such as bovine fetal serum and bovine calf serum, isgenerally used. Bovine fetal serum or bovine calf serum is in generalneeded to add to the basal medium at a level of about 5-20% by volume.

[0005] However, the serum component such as bovine fetal serum andbovine calf serum is limited in its supply and generally expensive. Thiswill increase manufacturing cost of a product of interest. Also, serumtends to be different in its characteristics between lots, which is notdesirable for culture that requires reproducibility. Further, it issometimes difficult to purify a product from a culture supernatant of aculture medium containing the serum. Furthermore, safety of the productof interest may not be sufficiently guaranteed since there is apossibility that serum derived from animals may be infected with prionsassociated with mad cow disease, which is feared to causeCreutzfeldt-Jacob disease, and scrapie in sheep, as well as viruses.

[0006] Furthermore, when a medium containing serum is used in anexperiment in the field of life science, the experimental system tendsto become complicated, which may result in problematic confusion upondiscussion of a causal relationship between cause and effect. This isbecause since serum comprises an extremely large variety of componentsincluding unknowns.

[0007] Therefore, attention has been drawn to cell culture media thatcontain known cell growth factors, hormones or the like instead of serumsuch as bovine fetal serum and bovine calf serum.

[0008] However, these cell growth factors or hormones are generally evenmore expensive than bovine fetal serum and bovine calf serum becausetheir presence in nature is scarce, which limits their use.

[0009] Accordingly, there is a need for safe and relatively inexpensivecell growth factors or cell growth means, which can replace theabovementioned serum, known cell growth factors and the like.

[0010] In general, animal culture cells are classified into adhesivecells and suspension cells depending on their state in culture.Discussion has been made on stimulation of cell growth in animal cellmass culture by contriving appropriate means that suit to the state ofcells in culture. For example, for adhesive cell culture, an attempt hasbeen made to stimulate the cell growth by increasing adhesivity of thecells. Here, in order to increase the cell adhesivity, use of thincoating with collagen on the surface of a material of a cell culture bedhas been discussed. However, the abovementioned risk of mad cow diseaseinfection has to be contemplated since collagen is generally derivedfrom cow, though it is relatively readily available.

[0011] Instead of the collagen-film coating treatment, use of a cellculture bed comprising silk film is suggested in Japanese PatentLaid-Open No. 11(1999)-243948 and Japanese Patent Laid-Open No.11(1999)-253155. Such a culture bed can increase cell adhesivity andstimulate cell growth like the abovementioned collagen-coated bed;however, handling becomes complicated since a crystallization process isrequired to make silk protein insoluble upon film formation.

[0012] Thus, use of silk-derived components for a culture bed has beenknown, but use of components derived from cocoons, raw silk or the likefor cell culture has not been discussed as far as the present inventorsare aware.

SUMMARY OF THE INVENTION

[0013] The present inventors have now found that animal cells ofinterest can efficiently grow when the animal cells are cultured in abasal medium for animal cell culture supplemented with sericin which canbe prepared from silkworm cocoons or the like. Further, such growthstimulating effect on animal cells by sericin has been similarlyobserved with chemically synthesized sericin and sericin obtained by agene engineering method. The present invention has been made based onthese findings.

[0014] Accordingly, it is an object of the present invention to providea medium supplement for animal cell culture, which can render theexcellent cell growth stimulating ability to a medium and is excellentin terms of safety and handling, and a medium for animal cell culture.

[0015] The medium supplement for animal cell culture according to thepresent invention comprises sericin or a sericin derivative.

[0016] Furthermore, the medium for animal cell culture according to thepresent invention comprises at least the abovementioned mediumsupplement and a basal medium composition.

[0017] According to another embodiment of the present invention, thereis provided a method of culturing animal cells, which comprises thesteps of adding the abovementioned medium supplement to an animal cellculture medium, culturing animal cells using the resulting medium, andgrowing said animal cells.

[0018] According to still another embodiment of the present invention,there is provided a method of producing a protein of interest, whichcomprises the steps of adding the abovementioned medium supplement to ananimal cell culture medium, culturing animal cells capable of producingthe protein using the resulting medium, and recovering the producedprotein from said medium and/or said animal cells.

[0019] According to yet still another embodiment of the presentinvention, there is provided a method of replicating a virus vector ofinterest, which comprises the steps of adding the abovementioned mediumsupplement to an animal cell culture medium, culturing animal cellsinfected with the virus vector using the resulting medium for growth,and recovering the virus vectors from said medium and/or said animalcells.

[0020] According to another embodiment of the present invention, thereis provided use of sericin or a sericin derivative for producing ananimal cell growth stimulating agent.

[0021] The medium supplement according to the present invention and themedium containing the same can stimulate the growth of animal cells tobe cultured and improve viability of the cells. Further, the productionof a useful substance can be promoted by applying them in culturinganimal cells capable of producing the useful substance of interest.Furthermore, such effects by the use of the medium supplement accordingto the present invention and the medium containing the same can berendered independently of the state of the cells, i.e., suspension cellsor adhesive cells, and the type of the cells, i.e., cell lines or normalcells.

[0022] Further, according to the present invention, safety of cultureproducts can be enhanced since the amount of a serum component such asbovine fetal serum and bovine calf serum can be reduced or its use canbe eliminated in culturing animal cells. The medium supplement accordingto the present invention is advantageous in terms of preparation andhandling since cell growth can be stimulated simply by adding it to amedium. Further, sericin used in the present invention can reduce costsfor production of animal cells and useful substances since it is lessexpensive than serum components.

DETAILED DESCRIPTION OF THE INVENTION

[0023] Medium Supplement

[0024] A medium supplement for culturing animal cells according to thepresent invention comprises sericin or a sericin derivative. Accordingto a preferred embodiment of the present invention, the mediumsupplement is used as an animal cell growth stimulating agent.

[0025] Sericin or Sericin Derivative

[0026] Sericin or a sericin derivative in the present invention can beeither naturally derived or artificially synthesized using ordinarychemical and/or genetic engineering methods, and either of them can beincluded.

[0027] Sericin

[0028] Sericin in the present invention implies any sericin protein allor a part of which is known to be naturally derived or synthesized. Thissericin has cell growth stimulating activity.

[0029] In the present invention, naturally derived sericin is preferablyobtained by a method described hereinafter.

[0030] In general, several kinds of sericin genes of 2.6 kbp to 10.6 kbpin length are confirmed and described, for example, in The Journal ofBiological Chemistry 257, 15192-15199 (1982). According to one preferredembodiment of the present invention, sericin has such gene sequences.

[0031] According to one preferred embodiment of the present invention,the entire sequence of sericin essentially comprises the amino acidsequence of SEQ ID NO: 2. Typically, this amino acid sequence comprisesan essential region consisting of 38 amino acids (SEQ ID NO: 1) andother nonessential regions. Preferably, sericin comprises a sequence ofmultiple repeats of the abovementioned essential region. For example,sericin comprising the amino acid sequence of SEQ ID NO: 2 contains 12repeats of the essential region consisting of 38 amino acids (SEQ ID NO:1).

[0032] Data for sericin base sequences encoding the amino acid sequenceof SEQ ID NO: 2 are registered at the EMBL data library with anaccession number: Z48802 and can be searched and available from NCBIhome page (http://www.ncbi.nlm.nih.gov/) or the like.

[0033] In this specification, the expression that “sericin essentiallycomprises the amino acid sequence represented by SEQ ID NO: 2” meansthat one or more (preferably 1 to 2000, more preferably 1 to 500, andstill more preferably 1 to 300) of amino acid residues in the amino acidsequence (SEQ ID NO: 2) can be deleted, substituted, inserted or added,as long as the sericin has cell growth stimulating activity. Accordingto a preferred embodiment, when the sericin is naturally derived, thenumber of amino acid residues deleted, substituted, inserted or added inthe abovementioned amino acid sequence can be preferably 1 to 2000, morepreferably 1 to 500, and still more preferably 1 to 300.

[0034] When the abovementioned deletions, substitutions, insertions oradditions of amino acid residues are present, they are preferablylocated in regions other than the essential region. In this case,conservative substitutions may be placed in the essential region.

[0035] According to a more preferred embodiment of the presentinvention, for example, one or more, preferably 1 to 50, and morepreferably 1 to 20 residues of the abovementioned amino acid sequence ofsericin can be conservatively substituted as long as the sericin hascell growth stimulating activity.

[0036] The term “conservative substitution” herein means substitution ofone or more amino acid residues with other chemically homologous aminoacid residues substantially without changing protein activity. Forexample, a certain hydrophobic residue can be substituted with anotherhydrophobic residue, a certain polar residue can be substituted withanother polar residue having the same charge, or a certain aromaticamino acid can be substituted with another aromatic amino acid.Functionally homologous amino acids which can be conservativelysubstituted in such a manner are known in the art for every amino acid.The following six groups are specific examples. These amino acids in thesame group can be conservatively substituted with each other.

[0037] (1) Alanine (Ala), serine (Ser) and threonine (Thr)

[0038] (2) Aspartic acid (Asp) and glutamic acid (Glu)

[0039] (3) Asparagine (Asn) and glutamine (Gln)

[0040] (4) Arginine (Arg) and lysine (Lys)

[0041] (5) Isoleucine (Ile), leucine (Leu), methionine (Met), and valine(Val), and

[0042] (6) Phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp).

[0043] In this specification, sericin naturally implies unhydrolyzedsericin (herein occasionally referred to as “sericin unhydrolysate”) butalso implies sericin hydrolysate. Here this sericin hydrolysate can beobtained by an ordinary method, for example, by hydrolyzing sericinusing acid, alkali, enzyme or the like.

[0044] Sericin Derivative

[0045] In the present invention, a sericin derivative is a polypeptidecomprising at least the amino acid sequence of SEQ ID NO: 1 consistingof 38 amino acids as an essential region. Preferably, this sericinderivative comprises the abovementioned essential region and anonessential region(s) at its one or both ends and has cell growthstimulating ability.

[0046] The expression “having cell growth stimulating ability” hereinmeans that cell growth stimulating activity of a polypeptide isrecognized by the skilled in the art. For example, it means such a casethat cell growth stimulating activity is recognized when measured underthe same conditions as described in Evaluation test 1 in Examplehereinafter.

[0047] Thus, the sericin derivative of the present invention maycomprise an essential region only or may comprise at least the essentialregion and any nonessential region other than the essential region, aslong as said sericin derivative has cell growth stimulating ability.

[0048] According to one preferred embodiment of the present invention,the sericin derivative has an amino acid sequence having the totallength of less than 2000, more preferably less than 500, and still morepreferably less than 300 residues.

[0049] Further, according to a more preferred embodiment of the presentinvention, the number of amino acid residues in the nonessentialregion(s) which can be present at one or both ends of the essentialregion is preferably less than 1000, more preferably less than 300, andstill more preferably less than 100.

[0050] According to a preferred embodiment of the present invention, thesericin derivative has a sequence of several repeats of theabovementioned amino acid sequence of SEQ ID NO: 1. Namely, the sericinderivative comprises one or more amino acid sequences of SEQ ID NO: 1other than the essential region. It is believed that a polypeptidehaving such a repetitive sequence has an improved cell growthstimulating activity.

[0051] According to a more preferred embodiment of the presentinvention, the sericin derivative has at least one, still morepreferably at least two amino acid sequences of SEQ ID NO: 1 per 100amino acid residues in its amino acid sequence. A high ratio of theamino acid sequence of SEQ ID NO: 1 contained in the sericin derivativeis preferable. Stable cell growth stimulating activity can be attainedby having repetitive sequences at such a ratio, even if the number ofamino acid residues in the total length of the sericin derivative isincreased.

[0052] When obtained by synthesis, the sericin derivative is preferably2 to 8 repeats, more preferably 2 to 6 repeats, and still morepreferably 2 to 4 repeats of the amino acid sequence of SEQ ID NO: 1.These figures are preferable because they are advantageous in syntheticproduction.

[0053] In the present invention, for example, one to several, preferably1 to 5, more preferably 1 to 3 amino acid sequences can beconservatively substituted in the essential region of the sericinderivative.

[0054] In the present invention, the sericin derivative includes afusion protein in which, for example, a polypeptide consisting of theabovementioned amino acid sequence of SEQ ID NO: 1 and a heterologouspolypeptide (for example, another functional protein) are combined.

[0055] In this specification, the sericin derivative naturally impliesan unhydrolyzed sericin or sericin derivative (herein occasionallyreferred to as “unhydrolysate”) and further implies a hydrolysate of thesericin derivative. This hydrolysate can be obtained by hydrolyzing thesericin derivative by an ordinary method, for example, using acid,alkali, enzyme or the like.

[0056] Naturally Derived Sericin or Sericin Derivative

[0057] According to one preferred embodiment of the present invention,the sericin and the sericin derivative can be derived from naturalsources. Naturally derived products are advantageous because they arehighly safe to the human body and relatively inexpensive. Such sericinor a sericin derivative can be used most appropriately as a mediumsupplement.

[0058] According to one preferred embodiment of the present invention,the sericin or the sericin derivative is extracted from cocoons or rawsilk. The cocoon means silkworm cocoon and the raw silk means silkfibers from silkworm cocoons.

[0059] In the present invention, the sericin or the sericin derivative,in particular their unhydrolysate, can be obtained from cocoons or rawsilk by an ordinary extracting method. More specifically, for example,it may be obtained by extraction as a highly purified single protein ata purity of greater than 90% as follows.

[0060] First, cocoons or raw silk is treated in water, preferably in hotwater at about 80-100° C. to solubilize sericin contained in the cocoonsor the raw silk in the water and thus an aqueous sericin solution isobtained. The aqueous sericin solution thus obtained is treated forisolation and purification, for example, using any of the followingmethods (1), (2) and (3) to recover the sericin unhydrolysate ofinterest.

[0061] (1) The pH of the aqueous sericin solution is adjusted to 3-5with an organic acid or inorganic acid, after which an organic coagulantor inorganic coagulant is added to precipitate sericin, and afterfiltration and drying, solid sericin is obtained.

[0062] (2) The aqueous sericin solution and a water soluble solvent suchas methanol, ethanol and dioxane are mixed to precipitate sericin, andafter filtration and drying, solid sericin is obtained.

[0063] (3) As described in Japanese Patent Laid-Open No.04(1992)-202435, the aqueous sericin solution is applied to anultrafiltration membrane or a reverse osmosis membrane, then specifiedfiltration treatment is carried out, and after drying, a sericin powderis obtained.

[0064] Further in the present invention, the hydrolysate of the sericinor the sericin derivative can be obtained from cocoons or raw silk by anordinary extraction method. More specifically, for example, it may beobtained by extraction as a highly purified single protein at a purityof greater than 90% as follows.

[0065] First, cocoons or raw silk is treated in water, preferably in hotwater at about 80-100° C. to solubilize sericin contained in the cocoonsor raw silk in the water and thus an aqueous sericin solution isobtained. At this stage, if necessary, sericin can be partly hydrolyzedusing electrolyzed water, acid, alkali or enzyme in combination. Theaqueous sericin solution thus obtained is treated for isolation andpurification, for example, using the abovementioned method (1), (2) or(3) to recover the sericin hydrolysate of interest.

[0066] In the present invention, preferably, the naturally derivedsericin or sericin derivative has a molecular weight distribution of500-500,000 and contains serine at a level of 20-40% by mol as an aminoacid.

[0067] Sericin or Sericin Derivative Obtained by Chemical or GeneticEngineering Method

[0068] According to one preferred embodiment of the present invention,sericin and a sericin derivative can be artificially synthesized usingan ordinary chemical or genetic engineering method. Typically, cellgrowth stimulating ability of such sericin or a sericin derivative isequal to or higher than naturally derived one. Accordingly, such sericinor a sericin derivative can also be suitably used as a mediumsupplement. Such chemical and genetic engineering methods for synthesiscan be appropriately used in combination, if necessary.

[0069] In the present invention, the sericin and the sericin derivativecan be a sequence which is all chemically synthesized or they can beobtained by using a partial sequence of a naturally derived sericin andfurther synthesizing based on the partial sequence. For chemicalsynthesis, an ordinary peptide synthesizing method such as a solidphase-liquid phase synthesis method using t-Boc method or Fmoc methodcan be appropriately used.

[0070] In the present invention, the sericin and the sericin derivativecan be produced by a genetic engineering method. Therefore, in thepresent invention, when a DNA encoding the sericin or the sericinderivative is available or can be constructed, the sericin and thesericin derivative can be produced in transformed cells obtained bytransforming host cells with such a DNA.

[0071] The DNA encoding a sericin-derived peptide can be obtained, forexample, by cloning from the silkworm silk gland, by chemical synthesis,or by using a partial DNA obtained from the silkworm silk gland andfurther synthesizing based on this partial DNA. Given an amino acidsequence of a peptide, a base sequence encoding, it can be in generaleasily determined referring to the so-called codon table. Accordingly,the DNAs encoding the sericin and the sericin derivative imply all thebase sequences having any degenerative codons.

[0072] The sericin and the sericin derivative can be each produced byobtaining a DNA, in particular in a form of a recombinant vector, whichcarries a DNA fragment encoding the sericin or the sericin derivative inan expressible state and is replicable in a host cell, transforming thehost cell using the DNA or the vector and culturing the transformantthus obtained. Namely, the so-called host-vector system can be used forthe production of said peptide. In applying such a host-vector system,various methods for constructing expression vectors (recombinantvectors) and transformation methods commonly used in the art can beused.

[0073] Vectors used for the production of the sericin and the sericinderivative can be selected from conventional vectors for which thehost-vector system has been established, such as plasmids, viruses,phages, and cosmid vectors, taking the kind of the host cell to be usedinto consideration. More specifically, for example, pBR, pUC or pQEplasmids or lambda-phage bacteriophage is used when Escherichia coli isused as a host cell, pUB plasmids can be used for Bacillus subtilis, andYEp and YCp vectors can be used for yeasts. Plasmids are preferably usedas a vector for producing said peptide.

[0074] A usable plasmid preferably contains a selective marker to selecttransformants. For example, a drug resistance marker, such as theampicillin resistance and kanamycin resistance markers, or a marker genefor auxotrophy can be used as such a selective marker. Further, recoveryof β-galactosidase activity by a specific peptide, which is produced bya vector DNA such as a plasmid, and a peptide encoded in a host cell canalso be used as a selective marker.

[0075] Further, the DNA as a recombinant vector preferably has DNAsequences necessary to express the sericin or the sericin derivative,for example, a promoter, transcription regulatory signals such as atranscription initiation signal, translation stop signal andtranscription termination signal, and translation regulatory signal.

[0076] Any cells can be used as a host cell for the production of thesericin and the sericin derivative as long as the host-vector system hasbeen established. Examples of such a host cell include Escherichia coli,Bacillus subtilis, yeasts and fungi.

[0077] When Bacillus subtilis, yeast or fungus is used as a host cell, asecretion type vector can be used as a vector to extracellularly excretethe sericin or the sericin derivative of interest.

[0078] Further, in the present invention, the sericin derivative can bein the form of a fusion protein. Such a fusion protein is produced byconstructing a DNA encoding the fusion protein by combining a DNAencoding a polypeptide comprising the abovementioned essential regionand a DNA encoding a heterologous polypeptide and then expressing theDNA thus constructed.

[0079] In this specification, the terms “DNA” and “gene” areoccasionally used interchangeably.

[0080] Medium for Culturing Animal Cells

[0081] An animal cell culture medium according to the present inventioncomprises at least the abovementioned medium supplement for animal cellculture medium and a basal medium composition. Accordingly, ifnecessary, it can contain various cell growth factors, for example,binding proteins such as albumin and transferrin, hormones such asinsulin, epithelial growth factor (EGF), fibroid cell growth factor andvarious steroid hormones, and cell adhesive factors such as fibronectin,as well as serum, as long as the abovementioned components are included.

[0082] According to a preferred embodiment of the present invention, theanimal cell culture medium is preferably a medium which contains serumin a smaller amount than ordinary media, and more preferably aserum-free medium. The serum-free medium is a medium which contains noserum and may contain cell growth factors and hormones other than serum.

[0083] The amount of the sericin or the sericin derivative contained inthe animal cell culture medium is not particularly limited, and can beappropriately changed depending on the kind of cells to be cultured, thepurpose of the culture, the kind of the basal medium composition and thelike.

[0084] According to a preferred embodiment of the present invention, thepercentage of the sericin or the sericin derivative in the medium is0.001-10% by weight, more preferably 0.02-0.5% by weight, and still morepreferably 0.05-0.2% by weight.

[0085] The present invention exhibits a sufficient effect even when asmall amount of the sericin or the sericin derivative is contained inthe medium of the present invention. However, even if they are added ina large amount, there would be generally no substantial problem sincesericin is nontoxic and highly water soluble.

[0086] When the medium supplement according to the present invention isadvantageously used by adding it to an ordinary medium, it is desirableto dissolve the medium supplement in a small volume of the medium andthen add it to the whole medium.

[0087] In the present invention, the basal medium composition comprisescarbon sources assimilatable by general animal cells, digestiblenitrogen sources and inorganic salts. More specifically, for example,inorganic salts, amino acids, glucose, and vitamins are included. Ifnecessary, a trace substance for nutritional stimulation and aneffective trace substance such as a precursor can be included in thebasal medium composition.

[0088] Any medium composition known to the skilled in the art can beused as such a basal medium composition. More specifically, for example,MEM medium (H. Eagle, Science, 130, 432 (1959)), DMEM medium (R.Dulbecco, Virology, 8, 396 (1959)), RPMI 1640 medium (G. E. Moore,J.A.M.A., 199, 519 (1967)), Ham's F12 medium (R. G. Ham, Proc. Natl.Acad. Sci. U.S.A., 53, 288 (1965)), MCDB104 medium (W. L. Mckeehan, InVitro, 13, 399 (1977)), and MCDB153 medium (D. M. Peehe, In Vitro, 16,526 (1980)) can be used.

[0089] Other media which can be appropriately used in the presentinvention include serum-free medium ASF104 (Ajinomoto Co., Inc.),serum-free medium SF-02 (Sanko Junyaku Co., Ltd.), serum-free mediumHybridoma-SFM (Lifetech Oriental), serum-free medium BIO-MPM-1(Biological Industries), serum-free medium EX-CELL™ 302-HDP (JRHBiosciences), serum-free medium Cosmedium 001 (Cosmo Bio), andserum-free medium SFM-101 (Nissui Pharmaceutical Co., Ltd.).

[0090] Animal cells which can be cultured in a medium of the presentinvention are not particularly limited and they can be eitherestablished cell lines or nonestablished normal cells obtained frombiological tissues. Accordingly, animal cells of the present inventioncan be, for example, cells which can produce proteins by themselves,cells which are transformed by genetic engineering to expressheterologous proteins, or cells which are infected with various virusvectors.

[0091] Examples of the cells which can produce proteins by themselvesinclude hybridoma cells producing monoclonal antibodies, leucocytesproducing interferon (IFN)-α, fibroblasts producing IFN-β, lymphocytesproducing IFN-γ, human kidney cells producing prourokinase (pro-UK) orUK, melanoma cells producing tissue plasminogen activator (tPA), In-111cells producing insulin, HIT cells producing glucagon, HepG2 cellsproducing erythropoietin, and B151K12 cells producing interleukin-5.

[0092] Examples of the cell lines transformed by genetic engineeringinclude Vero cells, HeLa cells, CHO (Chinese hamster ovary) cells, HKGcells, NIH3T3 cells, BHK cells, COS-1 cells, COS-7 cells, and myelomacells.

[0093] Examples of the cells infected with virus vectors include thoseinfected with retrovirus vectors, lentivirus vectors, adenovirusvectors, adeno-associated virus vectors, and herpesvirus vectors. Thesevirus vectors can be genetically recombined by an ordinary geneticengineering method. Further, examples of the animal cells which areinfected with these virus vectors and cultured using the medium of thepresent invention include HEK (human embryonic kidney) 293 cells, A549cells, and PER.C6 cells.

[0094] Another preferred embodiment of the present invention provides amethod of culturing animal cells, which comprises the steps of addingthe medium supplement of the present invention to an animal cell culturemedium and culturing animal cells using the resulting medium to grow theanimal cells.

[0095] Culture conditions for this method, for example, the oxygenconcentration, osmotic pressure, pH, temperature of the medium, can beappropriately changed depending on the kind of the cells to be cultured,the purpose of the culture, the volume of the culture, and the kind ofthe basal medium composition. Any culture system such as batch culture,continuous culture or perfusion culture can be used. High densityculture can also be used.

[0096] Still another preferred embodiment of the present inventionprovides a method of producing a protein, comprising the steps of addingthe medium supplement of the present invention to an animal cell culturemedium, culturing animal cells capable of producing the protein usingthe resulting medium to grow the animal cells, and recovering theproduced protein from said medium and/or said animal cells.

[0097] In the method of producing a protein according to the presentinvention, examples of the protein which can preferably be producedinclude monoclonal antibodies, IFN-α, IFN-β, INF-γ, pro-UK or UK, tPA,insulin, glucagon, erythropoietin, and interleukin-5.

[0098] The protein produced can be recovered using chemical or physicalcharacteristics of the protein and isolated and purified by variousordinary isolation methods. For example, the protein can be recovered,isolated and purified by treatment with a protein coagulant,ultrafiltration, absorption chromatography, ion-exchange chromatography,affinity chromatography, molecular sieving chromatography, dialysis orthe like, singly or in combination.

[0099] Another embodiment of the present invention provides a method ofreplicating a virus vector, which comprises the steps of adding themedium supplement of the present invention to an animal cell culturemedium, culturing to grow animal cells infected with the virus vectorsusing the resulting medium and recovering the produced virus vectorsfrom said medium and/or said animal cells.

[0100] Virus vectors replicable by the method of replication of thepresent invention are various virus vectors described above as examplesand can be created by genetic recombination, if necessary.

[0101] Appropriately selected animal cells are infected with the virusvectors of interest by an ordinary method.

[0102] Further, the virus vectors can be recovered from grown cells byisolation and purification using various ordinary isolation methods suchas ultrafiltration and centrifugation. Here it is desirable toappropriately select the method of recovering virus vectors according tothe kind of virus vectors.

[0103] Generally, gene therapies are categorized into two kinds, i.e.,ex vivo gene therapy and in vivo gene therapy. The former is atherapeutic method in which cells derived from a patient are firstcultured outside the body and then treated for gene transfer, afterwhich the cells are administered to the patient. The latter is atherapeutic method in which vectors with transferred genes are directlyintroduced into the patient's body.

[0104] The method according to the present invention can replicate virusvectors, into which genes used for such gene therapies are introduced,more efficiently than conventional methods. Further, the medium of thepresent invention exhibits excellent growth stimulating effect on theanimal cells used for such a replication method, such as 293 cells.

EXAMPLES

[0105] The present invention is further illustrated by the followingexamples that are not intended as a limitation of the invention.

[0106] Methods for producing sericin or a sericin derivative are shownin the following Production Example 1 and Production Example 2.

Production Example 1

[0107] One kilogram of cocoons (made by silkworms (Bombyx mori)) wastreated in 50 L of 0.2% sodium carbonate solution (pH 11-12) at 95° C.for 2 hours to extract sericin hydrolysate. The resulting sericinhydrolysate extract was filtered using a filter having an average porediameter of 0.2 μm to remove precipitate, after which the filtrate wasdesalted using a reverse osmosis membrane to obtain a transparent andcolorless aqueous solution of the sericin hydrolysate having a sericinconcentration of 0.2%.

[0108] This aqueous solution was concentrated using an evaporator to asericin concentration of about 2%, and then lyophilized to obtain 100 gof powdery sericin hydrolysate (polypeptide A) having an averagemolecular weight of 20,000 at a purity of greater than 90%.

Production Example 2

[0109] Chemical Synthesis of DNA Fragment Encoding Sericin Derivative:

[0110] A DNA was designed to encode a polypeptide, as one example of thesericin derivative, comprising the following amino acid sequenceincluding two repeats of the sequence (SEQ ID NO: 1) consisting of 38amino acids which is commonly conserved in sericin:Ser-Ser-Thr-Gly-Ser-Ser-Ser-Asn-Thr-Asp-Ser-Asn-Ser-Asn-Ser-Ala-Gly-Ser-Ser-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-Ser-Asn-Ser-Arg-Asp-Gly-Ser-Val-Ser-Ser-Thr-Gly-Ser-Ser-Ser-Asn-Thr-Asp-Ser-Asn-Ser-Asn-Ser-Ala-Gly-Ser-Ser-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-Ser-Asn-Ser-Arg-Asp-Gly-Ser-Val (SEQ ID NO: 4).

[0111] Here the recognition site (Ile-Glu-Gly-Arg (SEQ ID NO: 5)) ofprotease (Factor Xa) was placed at the N terminus of the abovementionedpeptide to cleave another fused peptide. Further, restriction enzymerecognition sites (PstI, EcoRI) were placed at both termini of the DNAencoding the abovementioned peptide to bind to a vector and twotranslation stop codons were added to the 3′ terminal side of the DNA.In this way, the DNA encoding the abovementioned polypeptide wasdesigned.

[0112] Next, the designed DNA was chemically synthesized using a DNAsynthesizer (Applied Biosystems) by the phosphoamidite method.Specifically, eight fragments each having a DNA length of about 60-70bases were chemically synthesized.

[0113] The synthesized eight DNA fragments were as follows: Fragment(1): 5′-GTGATCAATCGAAGGTCGCTCGAGTACTGGTTCTTCTTCTAACACCGACTCTAACTC (SEQID NO:6) TAAC-3′ Fragment (2):5′-TCTGCTGGTTCTTCTACCTCTGGTGGTTCTTCTACCTACGGTTACTCTTCTAACTCT (SEQ IDNO:7) CGTGACGGTTCT-3′ Fragment (3):5′-GTTTCTTCTACCGGTTCTTCTTCTAACACCGACTCTAACTCTAACTCTGCTGGTTCT (SEQ IDNO:8) TCTACCTC-3′ Fragment (4):5′-TGGTGGTTCTTCTACCTACGGTTACTCTTCTAACTCTCGTGACGGATCCGTTTAATA (SEQ IDNO:9) GCTGAGCG-3′ Fragment (1′):5′-CAGAGTTAGAGTTAGAGTCGGTGTTAGAAGAAGAACCAGTACTCGAGCGACCTTCGA (SEQ IDNO:10) TTGATCACTGCA-3′ Fragment (2′):5′-AAACAGAACCGTCACGAGAGTTAGAAGAGTAACCGTAGGTAGAAGAACCACCAGAGG (SEQ IDNO:11) TAGAAGAACCAG-3′ Fragment (3′):5′-ACCAGAGGTAGAAGAACCAGCAGAGTTAGAGTTAGAGTCGGTGTTAGAAGAAGAACC (SEQ IDNO:12) GGTAGAAG-3′ Fragment (4′):5′-AATTCGCTCAGCTATTAAACGGATCCGTCACGAGAGTTAGAAGAGTAACCGTAGGTA (SEQ IDNO:13) GAAGAACC-3′

[0114] Construction of DNA Encoding Peptide

[0115] The eight fragments (about 70 bases) synthesized as describedabove were converted into double-stranded chains by annealing withfragments each having a complementary sequence to obtain fourdouble-stranded DNA fragments consisting of DNA encoding the peptide.

[0116] Further, since a chemically synthesized oligonucleotide has nophosphoric acid at the 5′ terminus, phosphoric acid was added to the 5′terminus of each of the synthesized gene fragments using T4polynucleotide kinase (Takara Shuzo Co., Ltd.).

[0117] Next, the four DNA fragments were ligated using Takara LigationKit Version II (Takara Shuzo Co., Ltd.).

[0118] Construction of Expression Plasmid for Escherichia coli:

[0119] The ligated DNA fragment was mixed with a high expression vectorpQE 30 for Escherichia coli (Qiagen) and a ligation reaction was carriedout using Takara Ligation Kit Version II (Takara Shuzo Co., Ltd.). Theresulting reaction mixture was introduced into Escherichia coli JM109and then an expression plasmid, into which the DNA fragment wasinserted, was obtained from the transformant.

[0120] Expression Induction:

[0121]E. coli JM109 strain transformant cells, into which the expressionplasmid carrying the recombinant gene encoding the polypeptide wasintroduced, were cultured in M9+2% casamino acid medium supplementedwith 50 μg/ml ampicillin at 37° C. overnight with shaking. Afterculturing, the resulting culture was inoculated into the same medium ata concentration of 2%, and incubation was further continued at 37° C.with shaking.

[0122] IPTG (isopropyl-β-D-thiogalctopyranoside) at a finalconcentration of 1 mM was added to the resulting culture when theoptical density at 610 nm reached 0.3-0.5, and incubation was furthercontinued for 4 hours.

[0123] Purification of Peptide:

[0124] After the expression induction, the cells were broken byultrasonication and the resulting suspension was treated at 100° C. for10 minutes and centrifuged at 6,500 rpm for 5 minutes to recover thepolypeptide as a soluble fraction from the supernatant.

[0125] Next, the polypeptide was purified using QIA Express Ni-NTAProtein Purification System (Qiagen).

[0126] A sericin derivative (polypeptide B) having a molecular weight ofabout 8,000 was thus obtained by the method described above.

[0127] Evaluation Test

[0128] The following evaluation tests were carried out using the sericinand the sericin derivatives (polypeptides A and B) obtained in theProduction Examples above.

[0129] Evaluation Test 1: Stimulating Effect on Hybridoma Cell Growthand Stimulating Effect on Antibody Production

[0130] Sericin or bovine serum albumin (BSA) was added to and dissolvedin a serum-free basal medium ASF104 (Ajinomoto Co., Inc.) at a specifiedconcentration to prepare each of the media for 7 different experimentalgroups shown in Table 1 below.

[0131] Antibody producing mouse hybridoma cells (F. Makishima,Cytotechnology, 10, 15 (1992)) were inoculated at a cell density of1.5×10⁴ (cells/ml) into each of the media for experimental groupsprovided in wells of a 24-well culture plate. The hybridoma cells usedwere suspension cells.

[0132] Cells of each experimental group were cultured at 37° C. for 3days in an atmosphere of 5% CO₂ and 95% air by volume.

[0133] After culturing, cell density of the culture was measured usingan ordinary hemocytometer. Cell growth stimulating effect for eachexperimental group was evaluated from the change in the cell density.

[0134] The amount of antibodies produced in the culture supernatant wasquantitatively measured by an ordinary enzyme-linked immunosorbent assaymethod (ELISA method) using protein G purified antibody as a standard.In the measurement, horseradish peroxidase-labeled antimouse-IgG (G+L)was used as an enzyme-labeled secondary antibody, and o-phenylenediaminewas used as a color former. The amount of antibody in the cell-freeculture supernatant was expressed by the IgG concentration (μg/ml) in amedium. Antibody productivity in each experimental group was evaluatedfrom the change in antibody concentration.

[0135] Results obtained have been shown in Table 1 below. TABLE 1Antibody Cell density (10⁴ concentration Experimental group cells/ml)(μg/ml) No supplement added  8.2 ± 1.5 19 ± 2 Polypeptide A 0.05% 16.5 ±2.0 22 ± 2 Polypeptide A 0.1% 18.5 ± 1.3 23 ± 1 BSA 0.05% 15.5 ± 0.8 20± 1 BSA 0.1% 18.2 ± 1.7 18 ± 1 Polypeptide A and BSA, 23.4 ± 0.6 24 ± 20.05% each Polypeptide B 0.1% 22.3 ± 0.6 23 ± 2

[0136] These results showed that the media according to the presentinvention (each medium supplemented singly with polypeptide A orpolypeptide B) could strongly stimulate the growth and antibodyproductivity of the hybridoma cells. The effect was at least equal to orgreater than with the media supplemented with BSA.

[0137] Evaluation Test 2: Stimulating Effect on HepG2 Cell Growth,Enhancing Effect on Albumin Secretion and Stimulating Effect onSurvivability

[0138] Polypeptide A, polypeptide B or BSA was added to and dissolved ina serum-free medium SF-02 (Sanko Junyaku Co., Ltd.) at a specifiedconcentration to prepare each of the media for 6 different experimentalgroups shown in Table 2 below.

[0139] HepG2 cells derived from human liver cancer cells (J. Skelly,Nature, 282, 615 (1979)) were inoculated at a cell density of 1.0×10⁴(cells/ml) into wells of a 24-well culture plate. The HepG2 cells usedwere adhesive cells.

[0140] Cells of each experimental group were cultured at 37° C. in anatmosphere of 5% CO₂ and 95% air by volume.

[0141] After culturing, cell density of the culture was measured usingan ordinary hemocytometer. Cell growth stimulating effect for eachexperimental group was evaluated from the change in cell density.

[0142] Cell survivability was assessed by trypane blue staining andviable cell counting.

[0143] The amount of albumin secreted was quantitatively measured by anordinary enzyme-linked immunosorbent assay method (ELISA method) usingcommercial purified human serum albumin as a standard. In themeasurement, horseradish peroxidase-labeled antibody was used as anenzyme-labeled antibody, and o-phenylenediamine was used as a colorformer.

[0144] Results obtained have been shown in Table 2 below. TABLE 2 Celldensity of Secreted albumin Survivability Experimental culture at day 5in culture at day 5 in culture at group (10⁴ cells/ml) (ng/ml) day 17(%) No supplement 7.9 ± 0.6 390 ± 23 3.1 ± 0.5 added Polypeptide A 13.5± 0.4 431 ± 28 8.6 ± 0.4 0.05% Polypeptide A 17.3 ± 1.1 476 ± 19 10.3 ±0.8  0.1% BSA 0.05% 12.1 ± 0.8 420 ± 13 4.9 ± 0.4 BSA 0.1% 15.5 ± 0.7448 ± 24 6.2 ± 0.3 Polypeptide A 19.4 ± 1.2 510 ± 21 12.4 ± 0.7  andBSA, 0.05% each Polypeptide B 20.1 ± 1.5 525 ± 19 13.0 ± 0.4  0.1%

[0145] Average±standard deviation (n=3)

[0146] These results showed that the media according to the presentinvention strongly stimulated the growth of HepG2 cells and proteinproductivity characteristic to liver functions, such as albuminsecretion. Generally, when cells are cultured without changing a medium,most of the cells are killed due to overgrowth. In contract, cellsurvivability could be increased in the media according to the presentinvention.

[0147] Evaluation Test 3: Growth Stimulating Effect on Human EpidermalKeratinocyte Cells

[0148] Polypeptide A or B was added to and dissolved in a medium forepidermal keratinocyte cells (CCM-3111, Clonetics Corporation(California, USA)) at a specified concentration to prepare each of themedia for 4 different experimental groups shown in Table 3 below.

[0149] Neonatal epidermal keratinocyte cells (Normal Human EpidermalKeratinocyte cells, Clonetics Corporation (California, USA)) wereinoculated at a cell density of 1.0×10⁴ (cells/ml) into wells of a24-well culture plate. The cells used were normal cells and not cellline-derived and were adhesive cells.

[0150] Cells in each experimental group were cultured at 37° C. in anatmosphere of 5% CO₂ and 95% air by volume.

[0151] After culturing, cell density of the culture was measured usingan ordinary hemocytometer. Stimulating effect on cell growth for eachexperimental group was evaluated from the change in cell density.

[0152] Results obtained have been shown in Table 3 below. TABLE 3Experimental group Cell density (10⁴ cells/ml) No supplement added 3.4 ±0.1 Polypeptide A 0.01% 5.5 ± 1.3 Polypeptide A 0.05% 5.8 ± 0.8Polypeptide B 0.01% 5.9 ± 1.5 Polypeptide B 0.05% 6.3 ± 0.4

[0153] Average±standard deviation (n=3)

[0154] Occasionally, BSA cannot be added to a medium because cellsrather differentiate than proliferate when normal cells like epidermalkeratinocyte cells are cultured in a medium supplemented with BSA. Theresults above confirmed that the media according to the presentinvention stimulated the growth of epidermal keratinocyte cells and didnot induce cell differentiation.

[0155] Evaluation Test 4: Stimulating Effect on Adenovirus VectorProduction

[0156] This test was carried out using Takara Adenovirus ExpressionVector Kit (Takara Shuzo Co., Ltd.).

[0157] 4×10⁷ PFU of adenovirus was added to a medium (Nephrigen; Celox(Minnesota, USA)) containing 9.5×10⁵ of 293 cells and the admixture wasallowed to stand for one hour to infect the cells with the virus.

[0158] Next, the medium with cultured cells was replaced by 3 differentmedia for experimental groups shown in Table 4 below (one medium withoutsericin and two media each containing 0.02% polypeptide A or polypeptideB) and incubation was further continued for 3 days for viral production.Cells of each experimental group were cultured at 37° C. in anatmosphere of 5% CO₂ and 95% air by volume.

[0159] After culturing, the resulting virus suspension was recovered andthe virus titer was determined using 293 cells. The virus titer wascalculated by a TCID 50 method (50% tissue culture influence dose).

[0160] Results obtained have been shown in Table 4 below. TABLE 4Experimental group Virus titer (× 10⁷ PFU) No supplement added 3.36 ±1.50 Polypeptide A 0.02% added 5.18 ± 1.22 Polypeptide B 0.02% added7.62 ± 2.04

[0161] The results showed that the medium supplemented with sericin or asericin derivative according to the present invention stimulatedproduction of virus vector used for gene therapy. Such effect was highlymarked with polypeptide B.

1 13 1 38 PRT Artificial Sequence Peptides derived from sericin 1 SerSer Thr Gly Ser Ser Ser Asn Thr Asp Ser Asn Ser Asn Ser Ala 1 5 10 15Gly Ser Ser Thr Ser Gly Gly Ser Ser Thr Tyr Gly Tyr Ser Ser Asn 20 25 30Ser Arg Asp Gly Ser Val 35 2 1217 PRT Bombyx Mori 2 Met Arg Phe Val LeuCys Cys Thr Leu Ile Ala Leu Ala Ala Leu Ser 1 5 10 15 Val Lys Ala PheGly His His Pro Gly Asn Arg Asp Thr Val Glu Val 20 25 30 Lys Asn Arg LysTyr Asn Ala Ala Ser Ser Glu Ser Ser Tyr Leu Asn 35 40 45 Lys Asp Asn AspSer Ile Ser Ala Gly Ala Arg Arg Ala Lys Ser Val 50 55 60 Glu Gln Ser GlnAsp Lys Ser Lys Tyr Thr Ser Gly Pro Glu Gly Val 65 70 75 80 Ser Tyr SerGly Arg Ser Gln Asn Tyr Lys Asp Ser Lys Gln Ala Tyr 85 90 95 Ala Asp TyrHis Asn Asp Pro Asn Gly Gly Ser Ala Ser Ala Gly Gln 100 105 110 Ser ArgAsp Thr Ser Leu Arg Glu Arg Lys Val Asn Tyr Val Ser Asp 115 120 125 GlyGln Ala Val Ala Ala Ser Ser Asp Ala Arg Asp Glu Asn Arg Ser 130 135 140Ala Gln Gln Asn Ala Gln Ala Asn Trp Asn Ala Asp Gly Ser Tyr Gly 145 150155 160 Val Ser Ala Asp Arg Ser Gly Ser Ala Ser Ser Arg Arg Arg Gln Ala165 170 175 Asn Tyr Tyr Ser Asp Lys Asp Ile Thr Ala Ala Ser Lys Asp AspSer 180 185 190 Arg Ala Asp Ser Ser Arg Arg Ser Asn Ala Tyr Tyr Asn ArgAsp Ser 195 200 205 Asp Gly Ser Glu Ser Ala Gly Leu Ser Asp Arg Ser AlaSer Ser Ser 210 215 220 Lys Asn Asp Asn Val Phe Val Tyr Arg Thr Lys AspSer Ile Gly Gly 225 230 235 240 Gln Ala Lys Ser Ser Arg Ser Ser His SerGln Glu Ser Asp Ala Tyr 245 250 255 Tyr Asn Ser Ser Pro Asp Gly Ser TyrAsn Ala Gly Thr Arg Asp Ser 260 265 270 Ser Thr Ser Asn Lys Lys Lys AlaSer Ser Thr Ile Tyr Ala Asp Lys 275 280 285 Asp Gln Ile Arg Ala Ala AsnAsp Arg Ser Ser Ser Lys Gln Leu Lys 290 295 300 Gln Ser Ser Ala Gln IleSer Ser Gly Pro Lys Gly Thr Ser Val Ser 305 310 315 320 Ser Lys Asp ArgGln Tyr Ser Asn Asp Lys Arg Ser Lys Ser Asp Ala 325 330 335 Tyr Val GlyArg Asp Gly Thr Val Ala Tyr Ser Asn Lys Asp Ser Glu 340 345 350 Lys ThrSer Arg Gln Ser Asn Thr Asn Tyr Ala Asp Gln Asn Ser Val 355 360 365 ArgSer Asp Ser Ala Ala Ser Asp Gln Thr Ser Lys Ser Tyr Asp Arg 370 375 380Gly Tyr Ser Asp Lys Asn Ile Val Ala His Ser Ser Gly Ser Arg Gly 385 390395 400 Ser Gln Asn Gln Lys Ser Ser Ser Tyr Arg Ala Asp Lys Asp Gly Phe405 410 415 Ser Ser Ser Thr Asn Thr Glu Lys Ser Lys Phe Ser Ser Ser AsnSer 420 425 430 Val Val Glu Thr Ser Asp Gly Ala Ser Ala Ser Arg Glu SerSer Ala 435 440 445 Glu Asp Thr Lys Ser Ser Asn Ser Asn Val Gln Ser AspGlu Thr Gly 450 455 460 Glu Glu Glu Glu Leu Phe Asp Val Val Ser Tyr GlnLys Ile Glu Asp 465 470 475 480 Gly Lys Pro Val Ile Ile Met Lys Val IlePro Val Glu Lys Ser Ala 485 490 495 Ser Gln Ser Ser Ser Ser Arg Ser SerGln Glu Ser Ala Ser Tyr Ser 500 505 510 Ser Ser Ser Ser Ser Ser Thr LeuSer Glu Asp Ser Ser Glu Val Asp 515 520 525 Ile Asp Leu Gly Asn Leu GlyTrp Trp Trp Asn Ser Asp Asn Lys Ala 530 535 540 Gln Arg Ala Ala Gly GlyAla Thr Lys Ser Glu Ala Ser Ser Ser Thr 545 550 555 560 Gln Ala Thr ThrVal Ser Gly Ala Asp Asp Ser Ala Asp Ser Tyr Thr 565 570 575 Trp Trp TrpAsn Pro Arg Arg Ser Ser Ser Ser Ser Ser Ser Ala Ser 580 585 590 Ser SerSer Ser Gly Ser Asn Val Gly Gly Ser Ser Gln Ser Ser Gly 595 600 605 GlnSer Thr Ser Gly Ser Asn Ala Arg Gly His Leu Gly Thr Val Ser 610 615 620Ser Thr Gly Ser Thr Ser Asn Thr Asp Ser Ser Ser Lys Ser Ala Gly 625 630635 640 Ser Arg Thr Ser Gly Gly Thr Ser Thr Tyr Gly Tyr Ser Ser Ser His645 650 655 Arg Gly Gly Ser Val Ser Ser Thr Gly Ser Ser Ser Asn Thr AspSer 660 665 670 Ser Thr Lys Asn Ala Gly Ser Ser Thr Ser Gly Gly Thr SerThr Tyr 675 680 685 Gly Tyr Ser Ser Ser His Arg Gly Gly Ser Val Ser SerThr Gly Ser 690 695 700 Ser Ser Asn Thr Asp Ser Ser Thr Lys Ser Ala GlySer Ser Thr Ser 705 710 715 720 Gly Gly Thr Ser Thr Tyr Gly Tyr Ser SerArg His Arg Gly Gly Ser 725 730 735 Val Ser Ser Thr Gly Ser Ser Ser AsnThr Asp Ser Ser Thr Lys Asn 740 745 750 Ala Gly Ser Arg Thr Ser Gly GlyThr Ser Thr Tyr Gly Tyr Ser Ser 755 760 765 Ser His Arg Gly Gly Ser ValSer Ser Thr Gly Ser Ser Ser Asn Thr 770 775 780 Asp Ser Ser Thr Lys AsnAla Gly Ser Arg Thr Ser Gly Gly Thr Ser 785 790 795 800 Thr Tyr Gly TyrSer Ser Ser His Arg Gly Gly Ser Val Ser Ser Thr 805 810 815 Gly Ser SerSer Asn Thr Asp Ser Ser Thr Lys Asn Ala Gly Ser Arg 820 825 830 Thr SerGly Gly Thr Ser Thr Tyr Gly Tyr Ser Ser Ser His Arg Gly 835 840 845 GlySer Val Ser Ser Thr Gly Ser Ser Ser Asn Thr Asp Ser Ser Thr 850 855 860Lys Asn Ala Gly Ser Ser Thr Ser Gly Gly Ser Ser Thr Tyr Gly Tyr 865 870875 880 Ser Ser Asp Ser Arg Asp Gly Ser Val Ser Ser Thr Gly Ser Ser Ser885 890 895 Asn Thr Asp Ala Ser Thr Asp Leu Ala Gly Ser Ser Thr Ser GlyGly 900 905 910 Ser Ser Thr Tyr Gly Tyr Ser Ser Asp Ser Arg Asp Gly SerVal Ser 915 920 925 Ser Thr Gly Ser Ser Ser Asn Thr Asp Ala Ser Thr AspLeu Ala Gly 930 935 940 Ser Ser Thr Ser Gly Gly Ser Ser Thr Tyr Gly TyrSer Ser Asp Ser 945 950 955 960 Arg Asp Gly Ser Val Ser Ser Thr Gly SerSer Ser Asn Thr Asp Ala 965 970 975 Ser Thr Asp Leu Thr Gly Ser Ser ThrSer Gly Gly Ser Ser Thr Tyr 980 985 990 Gly Tyr Ser Ser Asp Ser Arg AspGly Ser Val Ser Ser Thr Gly Ser 995 1000 1005 Ser Ser Asn Thr Asp AlaSer Thr Asp Leu Ala Gly Ser Ser Thr 1010 1015 1020 Ser Gly Gly Ser SerThr Tyr Gly Tyr Ser Ser Ser Asn Arg Asp 1025 1030 1035 Gly Ser Val SerAla Thr Gly Ser Ser Ser Asn Thr Asp Ala Ser 1040 1045 1050 Thr Thr GluGlu Ser Thr Thr Ser Ala Gly Ser Ser Thr Glu Gly 1055 1060 1065 Tyr SerSer Ser Ser His Asp Gly Ser Val Thr Ser Thr Asp Gly 1070 1075 1080 SerSer Thr Ser Gly Gly Ala Ser Ser Ser Ser Ala Ser Thr Ala 1085 1090 1095Lys Ser Asp Ala Ala Ser Ser Glu Asp Gly Phe Trp Trp Trp Asn 1100 11051110 Arg Arg Lys Ser Gly Ser Gly His Lys Ser Ala Thr Val Gln Ser 11151120 1125 Ser Thr Thr Asp Lys Thr Ser Thr Asp Ser Ala Ser Ser Thr Asp1130 1135 1140 Ser Thr Ser Ser Thr Ser Gly Ala Ser Thr Thr Thr Ser GlySer 1145 1150 1155 Ser Ser Thr Ser Gly Gly Ser Ser Thr Ser Asp Ala SerSer Thr 1160 1165 1170 Ser Ser Ser Val Ser Arg Ser His His Ser Gly ValAsn Arg Leu 1175 1180 1185 Leu His Lys Pro Gly Gln Gly Lys Ile Cys LeuCys Phe Lys Asn 1190 1195 1200 Ile Phe Asp Ile Pro Tyr His Leu Arg LysAsn Ile Gly Val 1205 1210 1215 3 3651 DNA Bombyx Mori 3 atgcgtttcgttctgtgctg cactttgatt gcgttggctg cgctcagcgt aaaagctttc 60 ggtcaccaccccggcaatcg agatacagtc gaagtcaaaa accgaaagta caatgcagct 120 agcagtgaaagctcttacct caacaaagat aatgattcga taagtgccgg agcgcgccgt 180 gccaagtccgtagagcagag tcaggataaa agcaaatata catctggtcc agaaggcgtg 240 tcgtacagcggaaggtctca gaactataaa gattccaagc aagcttatgc cgattatcac 300 aacgatccgaacggcggatc tgcttctgcg ggacaatctc gcgacacgag cctgagggag 360 agaaaagtaaattacgtctc tgacggtcaa gcagtggccg cttccagtga cgctcgcgat 420 gaaaaccgatccgcccaaca gaatgctcag gccaattgga acgctgacgg ttcttacgga 480 gttagcgctgatcgaagtgg ttccgctagt tctagacgcc gccaagccaa ttactactcc 540 gataaagacatcactgctgc ttctaaagac gattcacgtg cagattcttc taggagaagc 600 aatgcctattacaacagaga tagtgacggc tcagaatccg ctggattaag tgaccgtagt 660 gcttcttcctcgaaaaatga taatgtattt gtttaccgca ctaaggattc tattggagga 720 caagcgaaatcttcaagatc atctcattca caagagagcg acgcttatta taactccagt 780 ccggatggaagctacaacgc tggtacgcga gacagttcaa cttctaacaa aaagaaggcg 840 agctctaccatctacgctga taaggatcaa atacgcgccg cgaatgatcg ttcttcttcg 900 aaacagttaaaacagagcag cgctcaaatc tcctccgggc caaagggcac ctctgtaagc 960 agtaaggataggcaatactc gaacgacaaa cgcagcaaat ctgatgcgta cgtcggacgg 1020 gacggcaccgttgcttactc aaacaaggac agcgaaaaga cctcacgaca aagtaatacg 1080 aactatgccgaccaaaactc cgttcgctct gactctgccg cttcggacca gaccagcaag 1140 agttacgacaggggctacag tgataaaaat atagttgccc atagctctgg tagtaggggc 1200 agtcagaatcagaaatcgtc gagttaccgc gctgacaagg acggtttttc ctccagtacg 1260 aatactgaaaaatccaaatt tagttcttcg aatagcgtcg tagaaacttc agatggagct 1320 tctgctagtcgcgaatcatc agcggaggat accaaatcat ccaatagtaa cgttcagagc 1380 gatgaaacaggcgaagaaga ggaattgttc gatgttgtat cttaccagaa aattgaagat 1440 ggcaagcctgtaatcataat gaaagttata ccagtcgaga aatccgcgtc ccaatcaagt 1500 tcttcgcggtcatctcagga gtctgcaagc tatagcagca gcagcagttc atcgacacta 1560 agtgaagactcttccgaggt ggatattgat cttggcaatt taggctggtg gtggaattca 1620 gacaataaggcacaaagagc ggcaggcggc gccacaaagt ctgaagcttc atcatccact 1680 caagctactacagtcagtgg cgcagacgac agtgctgatt cttacacctg gtggtggaat 1740 cctagacgatcaagcagctc ctcttcatca gcaagttcta gcagctctgg ctccaatgtt 1800 ggtggttcctctcaatccag cggtcagagc acttctggaa gtaatgcccg cggtcatcta 1860 ggaaccgtttcgtccactgg cagtaccagt aacaccgatt caagctcaaa aagtgcagga 1920 tcccgtacatccggcggtac gagcacttat ggatatagct ccagccatcg tggtggaagc 1980 gtatcatccaccggcagttc cagcaacact gattcaagca caaagaatgc aggatccagt 2040 acatctggcggtacgagcac ttatggatat agctctagcc atcgtggtgg aagtgtatca 2100 tccaccggcagttccagcaa cactgattca agcacaaaga gtgcaggatc cagtacatcc 2160 ggcggtacgagcacttacgg atatagctcc aggcatcgtg gtggaagcgt atcatccacc 2220 ggcagttccagcaacactga ttcaagcaca aagaatgcag gatcccgtac atccggcggt 2280 acgagcacttatggatatag ctccagccat cgtggtggaa gcgtatcatc caccggcagt 2340 tccagcaacactgattcaag cacaaagaat gcaggatccc gtacatccgg cggtacgagc 2400 acttatggatatagctccag ccatcgtggt ggaagcgtat catccaccgg cagttccagc 2460 aacactgattcaagcacaaa gaatgcagga tcccgtacat ccggcggtac gagcacttat 2520 ggatatagctccagccatcg tggtggaagc gtatcatcca ccggcagttc cagcaacact 2580 gattcaagcacaaagaatgc aggatccagt acatccggcg gtagcagcac ttatggatac 2640 agttccgacagtcgtgatgg aagtgtatca tccaccggca gttccagtaa cactgatgca 2700 agcacagacctggcaggatc cagtacatcc ggcggtagca gcacttatgg atacagttcc 2760 gacagtcgtgatggaagtgt atcatccacc ggcagttcca gtaacactga tgcaagcaca 2820 gacctggcaggatccagtac atccggcggt agcagcactt atggatacag ttccgacagt 2880 cgtgatggaagtgtatcatc caccggcagt tccagtaaca ctgatgcaag cacagacctt 2940 acaggatccagtacatccgg cggtagcagc acttatggat acagttccga cagtcgtgat 3000 ggaagtgtatcatccaccgg cagttccagt aacactgatg caagcacaga cctggcagga 3060 tccagtacatccggcggtag cagcacttat ggatatagct caagcaatcg tgatggaagt 3120 gtatcggccactggcagttc cagtaacact gatgcaagca ccacagaaga atccaccacg 3180 tccgctggtagcagcactga aggatatagt tccagtagcc atgatggaag cgtaacatcc 3240 accgacggttccagcacaag tggaggagct tcttccagct cagcgtcaac cgccaaaagc 3300 gacgccgcgtcatctgaaga cggtttctgg tggtggaata gaaggaaatc aggatccggt 3360 cacaaaagcgctaccgtaca gtcatccaca accgataaga cgagcaccga cagtgccagc 3420 agcaccgattccacctcaag cacgtccggg gcaagcacaa ccacttcagg cagttcttct 3480 acctcgggcggttcaagtac atcggacgct tcctccactt cgtctagtgt ttccaggagt 3540 catcattcaggcgtgaacag acttttacac aagcctggtc aaggaaaaat atgcctttgc 3600 ttcaaaaacatattcgatat tccttaccat ctccgtaaga atatcggtgt t 3651 4 76 PRT ArtificialSequence Peptides derived from sericin 4 Ser Ser Thr Gly Ser Ser Ser AsnThr Asp Ser Asn Ser Asn Ser Ala 1 5 10 15 Gly Ser Ser Thr Ser Gly GlySer Ser Thr Tyr Gly Tyr Ser Ser Asn 20 25 30 Ser Arg Asp Gly Ser Val SerSer Thr Gly Ser Ser Ser Asn Thr Asp 35 40 45 Ser Asn Ser Asn Ser Ala GlySer Ser Thr Ser Gly Gly Ser Ser Thr 50 55 60 Tyr Gly Tyr Ser Ser Asn SerArg Asp Gly Ser Val 65 70 75 5 4 PRT Artificial Sequence Recognitionsite for factor Xa 5 Ile Glu Gly Arg 1 6 61 DNA Artificial Sequence DNAfragment coded a part of the peptide derived from sericin 6 gtgatcaatcgaaggtcgct cgagtactgg ttcttcttct aacaccgact ctaactctaa 60 c 61 7 69 DNAArtificial Sequence DNA fragment coded a part of the peptide derivedfrom sericin 7 tctgctggtt cttctacctc tggtggttct tctacctacg gttactcttctaactctcgt 60 gacggttct 69 8 65 DNA Artificial Sequence DNA fragmentcoded a part of the peptide derived from sericin 8 gtttcttcta ccggttcttcttctaacacc gactctaact ctaactctgc tggttcttct 60 acctc 65 9 65 DNAArtificial Sequence DNA fragment coded a part of the peptide derivedfrom sericin 9 tggtggttct tctacctacg gttactcttc taactctcgt gacggatccgtttaatagct 60 gagcg 65 10 69 DNA Artificial Sequence DNA fragment codeda part of the peptide derived from sericin 10 cagagttaga gttagagtcggtgttagaag aagaaccagt actcgagcga ccttcgattg 60 atcactgca 69 11 69 DNAArtificial Sequence DNA fragment coded a part of the peptide derivedfrom sericin 11 aaacagaacc gtcacgagag ttagaagagt aaccgtaggt agaagaaccaccagaggtag 60 aagaaccag 69 12 65 DNA Artificial Sequence DNA fragmentcoded a part of the peptide derived from sericin 12 accagaggtagaagaaccag cagagttaga gttagagtcg gtgttagaag aagaaccggt 60 agaag 65 13 65DNA Artificial Sequence DNA fragment coded a part of the peptide derivedfrom sericin 13 aattcgctca gctattaaac ggatccgtca cgagagttag aagagtaaccgtaggtagaa 60 gaacc 65

1. A medium supplement for animal cell culture, comprising sericin or asericin derivative.
 2. The medium supplement according to claim 1,wherein the sericin derivative comprises at least a polypeptideconsisting of the amino acid sequence represented by SEQ ID NO: 1 as anessential region.
 3. The medium supplement according to claim 2, whereinthe sericin derivative comprises at least one polypeptide consisting ofthe amino acid sequence represented by SEQ ID NO: 1 per 100 amino acidresidues of said sericin derivative.
 4. The medium supplement accordingto claim 1, wherein the sericin derivative comprises 1 to 8 repeats ofthe amino acid sequence represented by SEQ ID NO:
 1. 5. The mediumsupplement according to claim 1, wherein the sericin or the sericinderivative is a polypeptide consisting of the amino acid sequencerepresented by SEQ ID NO: 2 or a polypeptide consisting of a modifiedamino acid sequence of SEQ ID NO: 2, in which one or more amino acidresidues are deleted, substituted, inserted or added, and having cellgrowth stimulating activity.
 6. The medium supplement according to claim1, wherein the sericin derivative is a fusion protein consisting of apolypeptide consisting of the amino acid sequence of SEQ ID NO: 1 and aheterologous protein.
 7. The medium supplement according to any one ofclaims 1 to 5, wherein the sericin or the sericin derivative isextracted from cocoons or raw silk.
 8. The medium supplement accordingto any one of claims 1 to 5, wherein the sericin or the sericinderivative is chemically synthesized.
 9. The medium supplement accordingto any one of claims 1 to 5, wherein the sericin or the sericinderivative is obtained by a genetic engineering method.
 10. The mediumsupplement according to any one of claims 1 to 9, which is used as ananimal cell stimulating agent.
 11. A medium for animal cell culture,comprising at least the medium supplement according to any one of claims1 to 10 and a basal medium composition.
 12. The medium according toclaim 11, which is a serum-free medium.
 13. The medium according toclaim 11, wherein the sericin or the sericin derivative is contained inthe medium at a level of 0.001-10% by weight.
 14. A method of culturinganimal cells, comprising the steps of adding the medium supplementaccording to claim 1 to an animal cell culture medium and culturinganimal cells using the resulting medium to grow said animal cells.
 15. Amethod of producing a protein of interest, comprising the steps ofadding the medium supplement according to claim 1 to an animal cellculture medium, culturing animal cells capable of producing the proteinof interest using the resulting medium, and recovering the producedprotein from said medium and/or said animal cells.
 16. A method ofreplicating a virus vector of interest, comprising the steps of addingthe medium supplement according to claim 1 to an animal cell culturemedium, culturing animal cells infected with the virus vector using theresulting medium for growth, and recovering the virus vectors from saidmedium and/or said animal cells.
 17. Use of sericin or a sericinderivative for producing an animal cell growth stimulating agent.